1-methylnicotinamide and its structural analog 1,4-dimethylpyridine for the prevention of cancer metastasis

Abstract

Background: 1-methylnicotinamide (1-MNA), an endogenous metabolite of nicotinamide, has recently gained interest due to its anti-inflammatory and anti-thrombotic activities linked to the COX-2/PGI2 pathway. Given the previously reported anti-metastatic activity of prostacyclin (PGI2), we aimed to assess the effects of 1-MNA and its structurally related analog, 1,4-dimethylpyridine (1,4-DMP), in the prevention of cancer metastasis.

Methods: All the studies on the anti-tumor and anti-metastatic activity of 1-MNA and 1,4-DMP were conducted using the model of murine mammary gland cancer (4T1) transplanted either orthotopically or intravenously into female BALB/c mouse. Additionally, the effect of the investigated molecules on cancer cell-induced angiogenesis was estimated using the matrigel plug assay utilizing 4T1 cells as a source of pro-angiogenic factors.

Results: Neither 1-MNA nor 1,4-DMP, when given in a monotherapy of metastatic cancer, influenced the growth of 4T1 primary tumors transplanted orthotopically; however, both compounds tended to inhibit 4T1 metastases formation in lungs of mice that were orthotopically or intravenously inoculated with 4T1 or 4T1-luc2-tdTomato cells, respectively. Additionally, while 1-MNA enhanced tumor vasculature formation and markedly increased PGI2 generation, 1,4-DMP did not have such an effect. The anti-metastatic activity of 1-MNA and 1,4-DMP was further confirmed when both agents were applied with a cytostatic drug in a combined treatment of 4T1 murine mammary gland cancer what resulted in up to 80 % diminution of lung metastases formation.

Conclusions: The results of the studies presented below indicate that 1-MNA and its structural analog 1,4-DMP prevent metastasis and might be beneficially implemented into the treatment of metastatic breast cancer to ensure a comprehensive strategy of metastasis control.

Keywords: 1,4-dimethylpyridinium chloride; 1-methylnicotinaimide chloride; Angiogenesis; Breast cancer; Combined therapy; Metastasis.

Fig. 1

Anti-metastatic efficacy of 1-MNA and 1,4-DMP in the model of experimental metastasis of 4T1-luc2-tdTomato cells. a Luminescence intensity of 4T1-luc2-tdTomato cells visualized during intravital imaging of mice treated with 1-MNA and 1,4-DMP (100 mg/kg/day in a drinking water) from the 7th day prior cancer cells intravenous transplantation. Data are presented as mean values ± SD. b Images of luminescence intensity taken on the 10th day of the experiment for untreated animals (upper row), mice treated with 1-MNA (middle row), mice treated with 1,4-DMP (bottom row). c Kaplan-Meier survival curves obtained for the animals treated with 1-MNA and 1,4-DMP (days of administration are indicated on the graph with an arrow)

Fig. 2

The influence of 1-MNA and 1,4-DMP on the growth, angiogenesis and metastasis of 4T1 tumors. a Tumor growth kinetics in mice treated with 1-MNA or 1,4-DMP given at the dose of 100 mg/kg/day in drinking water from the 7th day of the experiment (administration period is marked on the graph with an arrow). Data are presented as mean values ± SD. b Median number of lung metastases in BALB/c mice bearing 4T1 tumors. Data are presented as raw values with medians. c Wash-in-rate values estimated by the ultrasound imaging of 4T1 tumor blood perfusion performed on the 25th day of the experiment. Data are presented as median values ± min/max. d Representative images of tumor perfusion taken for: (I) control group, (II) mice treated with 1-MNA and (III) mice treated with 1,4-DMP

Fig. 3

The influence of 1-MNA and 1,4-DMP on tumor angiogenesis estimated in the matrigel plug assay. a Mean microvessel density (MVD) quantified in matrigel plug sections collected on the 7th day after their implantation and stained with anti-CD31 antibody. Data are presented as median values ± min/max. b Images of sections stained with anti-CD31 antibody for untreated animals (I), mice treated with cyclophosphamide (CP) at the dose of 25 mg/kg given intraperitoneally on days 0, 3 and 5 (II), mice treated with 1-MNA (III) and mice treated with 1,4-DMP (IV). Both 1-MNA and 1,4-DMP were given at the dose of 100 mg/kg/day in drinking water, starting from the day of matrigel plug implantation. Plasma levels of: c 6-keto-PGF1α, d TXB₂, e soluble P-selectin, f vWF in mice treated with cyclophosphamide, 1-MNA or 1,4-DMP; all data are presented as median values ± min/max, dotted lines on the graphs correspond to the value of the median concentration determined for respective agents in healthy BALB/c mice

Fig. 4

Anti-cancer efficacy of a combined treatment regime composed of 1-MNA or 1,4-DMP and cyclophosphamide in the 4T1 cancer model. For ease of interpretation, graphs are presented for the two therapeutic regimes separately. Tumor weight estimated on the 29^th day of the experiment for mice treated with a 1-MNA and cyclophosphamide (CP) or b 1,4-DMP and cyclophosphamide (CP) both presented with relevant controls; data are presented as median values  ± min/max. c Tumor growth inhibition (TGI) values observed for combined therapeutic regimes presented in reference to TGI of cyclophosphamide alone. In Fig. 4C, days of the drug administrations are indicated with arrows (1-MNA and 1,4-DMP - solid arrow, cyclophosphamide – gray arrows). The number of lung metastases in mice receiving d 1-MNA and cyclophosphamide (CP) or g 1,4-DMP and cyclophosphamide (CP) presented with corresponding controls; data are presented as raw values with medians. TXB₂ plasma levels in mice receiving e 1-MNA and cyclophosphamide (CP) or h 1,4-DMP and cyclophosphamide (CP); data are presented as median values ± min/max. vWF plasma levels in mice receiving f 1-MNA and cyclophosphamide (CP) or i 1,4-DMP and cyclophosphamide (CP); data are presented as median values ± min/max. j The images of bands obtained in Western blot analysis of tissue samples isolated from the animals assigned to treatment with: (a) control, (b) cyclophosphamide (CP), (c) 1-MNA, (d) 1-MNA and cyclophosphamide (CP), (e) 1,4-DMP, (f) 1,4-DMP and cyclophosphamide (CP). k E-cadherin : N-cadherin expression ratios in the samples of tumor tissue collected on the last day of the experiment. The total cellular content of E-cadherin (comprising protein characterized by the molecular weight of 100 and 130 kDa) and N-cadherin was first normalized to the content of β-actin and then used to determine E-cadherin to N-cadherin expression ratios that are presented on the graph as median values with min/max

Fig. 5

Body weight of BALB/c mice bearing 4T1 tumors, treated with a 1-MNA and cyclophosphamide (CP) or b 1,4-DMP and cyclophosphamide (CP), both presented with relevant controls. In Fig. 5a-b, days of the drug administrations are indicated with arrows (1-MNA or 1,4-DMP- solid arrow, cyclophosphamide – gray arrows). Data are presented as mean values ± SD